Bacillus thuringiensis cryIIIC, (b) protein toxic to coleopteran insects

ABSTRACT

A Bacillus thuringiensis strain isolate, designated EG5144, exhibits insecticidal activity against coleopteran insects, including Colorado potato beetle and insects of the genus Diabrotica. A novel toxin gene in B.t. strain EG5144 produces an irregularly shaped insecticidal crystal protein of approximately 70 kDa that is toxic to coleopteran insects. The cryIII-type gene (SEQ ID NO:1), designated as the cryIIIC(b) gene, has a nucleotide base sequence illustrated in FIG. 1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.08/032,775, filed Mar. 15, 1993, now U.S. Pat. No. 5,264,364, which is acontinuation of application Ser. No. 07/813,592, filed Dec. 23, 1991,now abandoned, which is a continuation-in-part

FIELD OF THE INVENTION

The present invention relates to an isolated Bacillus thuringiensisstrain, to its novel toxin encoding gene and to the insecticidal crystalprotein toxin made by the gene, as well as to insecticidal compositionscontaining the protein that are toxic to coleopteran insects.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (hereinafter "B.t.") is a gram-positive soilbacterium that produces crystal proteins during sporulation which arespecifically toxic to certain orders and species of insects. Manydifferent strains of B.t. have been shown to produce insecticidalcrystal proteins. Compositions including B.t. strains which produceinsecticidal proteins have been commercially available and used asenvironmentally acceptable insecticides because they are quite toxic tothe specific target insect, but are harmless to plants and othernon-targeted organisms.

A number of genes encoding crystal proteins have been cloned fromseveral strains of B.t. A review of such genes is set forth in H. Hofteet al., Microbiol. Rev., 53, pp.242-255 (1989). This reference providesa good overview of the genes and proteins obtained from B.t. and theiruses, adopts a nomenclature and classification scheme for B.t. genes andproteins, and has an extensive bibliography.

The B.t. crystal protein is toxic in the insect only after ingestion.After ingestion, the alkaline pH and proteolytic enzymes in the insectmid-gut solubilize the crystal allowing the release of the toxiccomponents. These toxic components disrupt the mid-gut cells causing theinsect to cease feeding and, eventually, to die. In fact, B.t. hasproven to be an effective and environmentally safe insecticide indealing with various insect pests.

As noted by Hofte et al., the majority of insecticidal B.t. strains areactive against insects of the order Lepidoptera, i.e., caterpillarinsects. Other B.t. strains are insecticidally active against insects ofthe order Diptera, i.e., flies and mosquitoes, or against bothlepidopteran and dipteran insects. In recent years, a few B.t. strainshave been reported as producing crystal protein that is toxic to insectsof the order Coleoptera, i.e., beetles.

The first isolation of a coleopteran-toxic B.t. strain is reported by A.Krieg et al., in Z.angew. Ent., 96, pp.500-508 (1983); see also A. Krieget al., Anz. Schaedlingskde., Pflanzenschutz, Umweltschutz, 57,pp.145-150 (1984) and U.S. Pat. No. 4,766,203, issued Aug. 23, 1988 ofA. Krieg et al. The strain, designated B.t. var. tenebrionis, isreported to be toxic to larvae of the coleopteran insects Agelasticaalni (blue alder leaf beetle) and Leptinotarsa decemlineata (Coloradopotato beetle). B.t. tenebrionis makes an insecticidal crystal proteinreported to be about 65-70 kilodaltons (kDa) (U.S. Pat. No. 4,766,203;see also K. Bernhard, FEMS Microbiol.Lett., 33, pp.261-265 (1986)).

V. Sekar et al., Proc. Natl. Acad. Sci. USA, 84, pp.7036-7040 (1987),report the cloning and characterization of the gene for thecoleopteran-toxic crystal protein of B.t. tenebrionis. The size of theprotein, as deduced from the sequence of the gene, was 73 kDa, but theisolated protein contained primarily a 65 kDa component. Hofte et al.,Nucleic Acids Res., 15, p.7183 (1987), also report the DNA sequence forthe cloned gene from B.t. tenebrionis, and the sequence of the gene isidentical to that reported by Sekar et al. (1987).

McPherson et al., Bio/Technology, 6, pp.61-66 (1988), disclose the DNAsequence for the cloned insect control gene from B.t. tenebrionis, andthe sequence is identical to that reported by Sekar et al. (1987). E.coli cells and Pseudomonas fluorescens cells harboring the cloned genewere found to be toxic to Colorado potato beetle larvae.

PCT International Publication No. WO 91/07481 dated May 30, 1991, ofNovo Nordisk A/S, describes B.t. mutants that produce high yields of thesame insecticidal proteins originally made by the parent strains atlesser yields. Mutants of the coleopteran-toxic B.t. tenebrionis strainare disclosed.

A coleopteran-toxic strain, designated B.t. var. san diego, is reportedby C. Herrnstadt et al., Bio/Technology, 4, pp.305-308 (1986), toproduce a 64 kDa crystal protein that was toxic to various coleopteraninsects: strong toxicity to Pyrrhalta luteola (elm leaf beetle);moderate toxicity to Anthonomus grandis (boll weevil), Leptinotarsadecemlineata (Colorado potato beetle), Otiorhynchus sulcatus (black vineweevil), Tenebrio molitor (yellow mealworm) and Haltica tombacina; andweak toxicity to Diabrotica undecimpunctata undecimpunctata (westernspotted cucumber beetle).

The DNA sequence of the cloned coleopteran toxin gene of B.t. san diegois reported in C. Herrnstadt et al., Gene, 57, pp.37-46 (1987); see alsoU.S. Pat. No. 4,771,131, issued Sep. 13, 1988, of Herrnstadt et al. Thesequence of the toxin gene of B.t. san diego is identical to thatreported by Sekar et al. (1987) for the cloned coleopteran toxin gene ofB.t. tenebrionis.

A. Krieg et al., J.Appl.Ent., 104, pp.417-424 (1987), report that thestrain B.t. san diego is identical to the B.t. tenebrionis strain, basedon various diagnostic tests.

Another new B.t. strain, designated EG2158, is reported by W. P. Donovanet al., in Mol.Gen.Genet., 214, pp.365-372 (1988) and in U.S. Pat. No.5,024,837 issued Jun. 18, 1991, to produce a 73 kDa crystal protein thatis insecticidal to coleopteran insects. The toxin-encoding gene fromB.t. strain EG2158 was cloned and sequenced, and its sequence isidentical to that reported by Sekar et al. (1987) for the cloned B.t.tenebrionis coleopteran toxin gene. This coleopteran toxin gene isreferred to as the cryIIIA gene by Hofte et al., Microbiol.Rev., 53,pp.242-255 (1989).

The Donovan et al. '837 U.S. patent noted above also describes hybridB.t. var. kurstaki strains designated EG2424 and EG2421, which areactive against both lepidopteran insects and coleopteran insects. Thebeetle activity of these hybrid strains results from the coleopterantoxin plasmid transferred from B.t. strain EG2158 by conjugal plasmidtransfer.

U.S. Pat. No. 4,797,279, issued Jan. 10, 1989, of D. Karamata et al.(corresponding to EP-A-0 221 024), discloses a hybrid B.t. microorganismcontaining a plasmid from B.t. var. kurstaki with a lepidopteran toxingene and a plasmid from B.t. tenebrionis with a coleopteran toxin gene.The hybrid B.t. produces crystal proteins characteristic of those madeby B.t. kurstaki, as well as those of B.t. tenebrionis.

U.S. Pat. No. 4,910,016, issued Mar. 20, 1990, of Gaertner et al.(corresponding to EP-A-0 303 379), discloses a novel B.t. isolateidentified as B.t. MT 104 which has insecticidal activity against twoorders of insects, Colorado potato beetle (Coleoptera) and cabbagelooper (Lepidoptera).

European Patent Application Publication No. 0 318 143, published May 31,1989, of Lubrizol Genetics, Inc., discloses the cloning,characterization and selective expression of the intact partiallymodified gene from B.t. tenebrionis, and the transfer of the cloned geneinto a host microorganism rendering the microorganism able to produce aprotein having toxicity to coleopteran insects. Insect bioassay data forB.t. san diego reproduced from Herrnstadt et al., Bio/Technology, 4,pp.305-308 (1986) discussed above, is summarized. The summary alsoincludes data for B.t. tenebrionis from another source; B.t. tenebrionisis reported to exhibit strong toxicity to Colorado potato beetle,moderate toxicity to western corn rootworm (Diabrotica virgifera) andweak toxicity to southern corn rootworm (Diabrotica undecimpunctata).

European Patent Application Publication No. 0 324 254, published Jul.19, 1989, of Imperial Chemical Industries PLC, discloses a novel B.t.strain identified as A30 which has insecticidal activity againstcoleopteran insects, including Colorado potato beetle larvae, cornrootworm larvae and boll weevils.

U.S. Pat. No. 4,999,192, issued Mar. 12, 1991, of Payne et al.(corresponding to EP A-0 328 383), discloses a novel B.t. microorganismidentified as B.t. PS40D1 which has insecticidal activity againstColorado potato beetle larvae. B.t. strain PS40D1 is identified viaserotyping as being serovar 8a 8b, morrisoni.

U.S. Pat. No. 5,006,336, issued Apr. 9, 1991, of Payne et al.(corresponding to EP-A-0 346 114), discloses a novel B.t. isolatedesignated as PS122D3, which is serotyped as serovar 8a8b, morrisoni andwhich exhibits insecticidal activity against Colorado potato beetlelarvae.

U.S. Pat. No. 4,966,765, issued Oct. 30, 1990, of Payne et al.(corresponding to EP-A-0 330 342), discloses a novel B.t. microorganismidentified as B.t. PS86B1 which has insecticidal activity against theColorado potato beetle. B.t. strain PS86B1 is identified via serotypingas being serovar tolworthi.

The nucleotide sequence of a cryIIIB gene and its encodedcoleopteran-toxic protein is reported by Sick et al., in Nucleic AcidsRes., 18, p.1305 (1990) but the B.t. source strain is identified onlyvia serotyping as being subspecies tolworthi. U.S. Pat. No. 4,966,155,issued Feb. 26, 1991, of Sick et al. (corresponding to EP-A-0 337 604),discloses a B.t. toxin gene obtained from the coleopteran-active B.t.strain 43F, and the gene sequence appears identical to the cryIIIB gene.B.t. strain 43F is reported as being active against Colorado potatobeetle and Leptinotarsa texana.

European Patent Application No. 0 382 990, published Aug. 22, 1990, ofPlant Genetic Systems N.V., discloses two novel B.t. strains bt PG51208and bt PG51245 producing respective crystal proteins of 74 and 129 kDathat exhibit insecticidal activity against Colorado potato beetlelarvae. The DNA sequence reported for toxin gene producing the 74 kDaprotein appears to be related to that of the cryIIIB gene of Sick et al.

PCT International Publication No. WO 90/13651, published Nov. 15, 1990,of Imperial Chemical Industries PLC, discloses novel B.t. strains whichcontain a toxin gene encoding an 81 kDa protein that is stated to betoxic not only to lepidopteran insects but also to coleopteran insects,including Diabrotica.

U.S. Pat. No. 5,055,293, issued Oct. 8, 1991, of Aronson et al.,discloses the use of B. laterosporous for corn rootworm (Diabrotica)insect control.

The various B.t. strains described in aforementioned literature arereported to have crystal proteins insecticidally active againstcoleopteran insects, but none has been demonstrated to have significant,quantifiable toxicity to the larvae and adults of the insect genusDiabrotica (corn rootworm), which includes the western corn rootworm(Diabrotica virgifera virgifera), the southern corn rootworm (Diabroticaundecimpunctata howardi) and the northern corn rootworm (Diabroticabarberi).

The B.t. strain of the present invention contains a novel toxin genethat expresses protein toxin having quantifiable insecticidal activityagainst the Diabrotica insects, among other coleopteran insects.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a purified and isolatedcoleopteran toxin gene having a nucleotide base sequence coding for theamino acid sequence illustrated in FIG. 1 and hereinafter designated asthe cryIIIC(b) gene (SEQ ID NO:1). The cryIIIC(b) gene (SEQ ID NO:1) hasa coding region extending from nucleotide bases 144 to 2099 shown inFIG. 1.

Another aspect of the present invention relates to the insecticidalprotein produced by the cryIIIC(b) gene. The CryIIIC(b) protein (SEQ IDNO:2) has the amino acid sequence, as deduced from the nucleotidesequence of the cryIIIC(b) gene (SEQ ID NO:1) from nucleotide bases 144to 2099 that is shown in FIG. 1. The protein exhibits insecticidalactivity against insects of the order Coleoptera, particular, Coloradopotato beetle and insects of the genus Diabrotica.

Still another aspect of the present invention relates to a biologicallypure culture of a B.t. bacterium deposited with the AgriculturalResearch Culture Collection, Northern Regional Research Laboratory(NRRL) having Accession No. NRRL B-18655 and being designated as B.t.strain EG5144 and a biologically pure culture of a second bacteriumdeposited with the NRRL having Accession No. NRRL B-18920 and beingdesignated as B.t. strain EG5145. B.t. strain EG5144 is a wild-type B.t.strain that carries the cryIIIC(b) gene (SEQ ID NO:1) and produces theinsecticidal CryIIIC(b) protein (SEQ ID NO:2). B.t. strain EG5145 isalso a wild-type B.t. strain, whose characteristics are similar to thoseof B.t. strain EG5144 described in more detail below. Biologically purecultures of other B.t. bacteria carrying the cryIIIC(b) gene (SEQ IDNO:1) are also within the scope of this invention.

Yet another aspect of this invention relates to insecticidalcompositions containing, in combination with an agriculturallyacceptable carrier, either the CryIIIC(b) protein (SEQ ID NO:2) orfermentation cultures of a B.t. strain which has produced the CryIIIC(b)protein.

The invention also includes a method of controlling coleopteran insectsby applying to a host plant for such insects an insecticidally effectiveamount of the CryIIIC(b) protein (SEQ ID NO:2) or of a fermentationculture of a B.t. strain that has made the CryIIIC(b) protein. Themethod is applicable to a variety of coleopteran insects, such as theColorado potato beetle, Japanese beetle larvae (white grubs), Mexicanbean beetle and corn rootworm.

Still another aspect of the present invention relates to a recombinantplasmid containing the cryIIIC(b) gene (SEQ ID NO:1), a biologicallypure culture of a bacterium transformed with such recombinant plasmid,the bacterium preferably being B.t. such as B.t. strain EG7237 describedin Example 6, as well as a plant transformed with the cryIIIC(b) gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises FIGS. 1A through 1C and shows the nucleotide basesequence of the cryIIIC(b) gene (SEQ ID NO:1) and the deduced amino acidsequence of the CryIIIC(b) protein (SEQ ID NO:2). The putative ribosomebinding site (RBS) is indicated. Restriction sites for SspI and HindIIIare also indicated.

FIG. 2 is a photograph of an ethidium bromide stained agarose gelcontaining size fractionated native plasmids of B.t. strains EG5144(lane 1), EG4961 (lane 2), EG2838 (lane 3) and EG2158 (lane 4). Thenumbers to the left of FIG. 2 indicate the approximate sizes, inmegadaltons (MDa), of the plasmids of B.t. strain EG5144.

FIG. 3 is a photograph of an autoradiogram made by transferring sizefractionated DNA fragments from an agarose gel to a nitrocellulosefilter, hybridizing the filter with a radioactively labeled 2.4kilobases (kb) cryIIIB probe, and exposing the filter to X-ray film. Theagarose gel contained size fractionated total DNA fragments from B.t.strains EG2158, EG5144, EG2838 and EG4961, that had been obtained inseparate digestions with the restriction enzymes SspI, HindIII andEcoRI. The numbers to the left of FIG. 3 indicate the sizes, in kb, ofB.t. strain EG5144 restriction fragments that hybridized to the cryIIIBprobe. The lane labeled "stnd" is a size standard.

FIG. 4 is a photograph of a Coomassie stained sodium dodecyl sulfate("SDS") polyacrylamide gel showing crystal proteins solubilized fromB.t. strains EG5144 (lane 1), EG4961 (lane 2), EG2158 (lane 3) andEG2838 (lane 4). The numbers to the left of FIG. 4 indicate theapproximate sizes in kDa of the crystal proteins produced by B.t. strainEG5144. Lane 5 contains protein molecular size standards.

FIG. 5 shows a restriction map of plasmid pEG271. The location andorientation of the cryIIIC(b) gene (SEQ ID NO:1) is indicated by thearrow. Plasmid pEG271 is functional in Escherichia coli (E. coli), sinceit contains Coli plasmid pUC18 (Ap^(r)), indicated by the segment markedpUC18. The abbreviations for the restriction endonuclease cleavage sitesare as follows: Ba=BamHI; Bg=Bg1II; H=HindIII; R=EcoRI; S=SphI; andX=XbaI. A one kilobase scale marker is also illustrated.

FIG. 6, aligned with and based on the same scale as FIG. 5, shows arestriction map of plasmid pEG272. The location and orientation of thecryIIIC(b) gene (SEQ ID NO:1) is indicated by the arrow shown in FIG. 5.Plasmid pEG272 is derived from plasmid pEG271 (FIG. 5) and contains theBacillus plasmid pNN101 (Cm^(r) Tc^(r)), indicated by the segment markedpNN101 and is incorporated into the SphI site of pEG271; this plasmid isfunctional in B.t. Abbreviations are the same as those for FIG. 5.

FIG. 7 is a photograph of a Coomassie stained SDS-polyacrylamide gel.The gel shows protein bands synthesized by B.t. strain EG5144 (lane 1)and by recombinant B.t. strain EG7237 containing pEG272 (lane 3). Lane 2contains a protein size standard and the numbers on either side of lanes1 and 3 indicate approximate sizes, in kDa, of the Crystal proteinsproduced by these strains.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The isolation and purification of the cryIIIC(b) gene (SEQ ID NO:1) andthe coleopteran-toxic CryIIIC(b) crystal protein (SEQ ID NO:2) and thecharacterization of the new B.t. strain EG5144 which produces theCryIIIC(b) protein are described at length in Examples 1-7. The utilityof B.t. strain EG5144 and of the CryIIIC(b) crystal protein (SEQ IDNO:2) in insecticidal compositions and methods is also illustrated inExamples 8-11.

The cryIII-type gene of this invention, the cryIIIC(b) gene (SEQ IDNO:1), has the nucleotide base sequence shown in FIG. 1. The codingregion of the cryIIIC(b) gene (SEQ ID NO:1) extends from nucleotide baseposition 144 to position 2099 shown in FIG. 1.

A comparison of the nucleotide base sequence of the cryIIIC(b) genecoding region with the corresponding coding region of the prior artcryIIIA gene indicates significant differences between the two genes.The cryIIIC(b) gene (SEQ ID NO:1) is only 76% homologous (positionallyidentical) with the cryIIIA gene.

A comparison of the nucleotide base sequence of the cryIIIC(b) genecoding region with the corresponding coding region of the cryIIIB geneobtained from recently discovered B.t. strain EG2838 (NRRL Accession No.B-18603) indicates that the cryIIIC(b) gene (SEQ ID NO:1) is 96%homologous (positionally identical) with the cryIIIB gene.

The CryIII-type protein of this invention, the CryIIIC(b) protein, thatis encoded by the cryIIIC(b) gene (SEQ ID NO:1), has the amino acidsequence (SEQ ID NO:2) shown in FIG. 1. In this disclosure, referencesto the CryIIIC(b) "protein" are synonymous with its description as a"crystal protein", "protein toxin", "insecticidal protein" or the like,unless the context indicates otherwise. The size of the CryIIIC(b)protein (SEQ ID NO:2), as deduced from the DNA sequence of thecryIIIC(b) gene (SEQ ID NO:1), is 74,265 Daltons (Da).

The size of the CryIIIB protein, as deduced from the sequence of thecryIIIB gene, is 74,237 Da. The prior art CryIIIA protein, encoded bythe cryIIIA gene, has a deduced size of 73,116 Da.

Despite the apparent size similarity, comparison of the amino acidsequence of the CryIIIC(b) protein (SEQ ID NO:2) with that of the priorart CryIIIA protein shows significant differences between the two. TheCryIIIC(b) protein (SEQ ID NO:2) is only 68% homologous (positionallyidentical amino acids) with the CryIIIA protein. The CryIIIC(b) protein(SEQ ID NO:2) is 95% homolgous with the CryIIIB protein. Nevertheless,despite the apparent homology of the CryIIIC(b) and CryIIIB proteins,the CryIIIC(b) protein (SEQ ID NO:2) has been shown to be a differentprotein than the CryIIIB protein, based on its significantly improvedinsecticidal activity compared to the CryIIIB protein with respect toinsects of the order Coleoptera and in particular, insects of the genusDiabrotica. The CryIIIC(b) protein (SEQ ID NO:2), unlike the CryIIIBprotein, exhibits quantifiable insecticidal activity against cornrootworm larvae.

The present invention is intended to cover mutants and recombinant orgenetically engineered derivatives, e.g., truncated versions, of thecryIIIC(b) gene (SEQ ID NO:1) that yield a protein with insecticidalproperties essentially the same as those of the CryIIIC(b) protein (SEQID NO:2).

The cryIIIC(b) gene (SEQ ID NO:1) is also useful as a DNA hybridizationprobe, for discovering similar or closely related cryIII-type genes inother B.t. strains. The cryIIIC(b) gene (SEQ ID NO:1), or portions orderivatives thereof, can be labeled for use as a hybridization probe,e.g., with a radioactive label, using conventional procedures. Thelabeled DNA hybridization probe may then be used in the manner describedin the Examples.

The cryIIIC(b) gene (SEQ ID NO:1) and the corresponding insecticidalCryIIIC(b) protein (SEQ ID NO:2) were first identified in B.t. strainEG5144, a novel B.t. isolate. The characteristics of B.t. strain EG5144are more fully described in the Examples. Comparison of the plasmidarrays and other strain characteristics of B.t. strain EG5144 with thoseof the recently discovered B.t. strains EG2838 and EG4961 and those ofthe prior art B.t. strain EG2158 and B.t. var. tenebrionis (or theequivalent, B.t. var. san diego) demonstrates that each of thesecoleopteran-toxic B.t. strains is distinctly different. The plasmidarray of B.t. strain EG5145, another wild-type strain isolated alongwith B.t. strain EG5144, is similar to that of B.t. strain EG5144, andB.t. strain EG5145 exhibits the same insecticidal activity againstcoleopteran insects, e.g., Japanese beetle larvae, as that of B.t.strain EG5144 (see Example 11).

The cryIIIC(b) gene (SEQ ID NO:1) may be introduced into a variety ofmicroorganism hosts, using procedures well known to those skilled in theart for transforming suitable hosts under conditions which allow forstable maintenance and expression of the cloned cryIIIC(b) gene.Suitable hosts that allow the cryIIIC(b) gene (SEQ ID NO:1) to beexpressed and the CryIIIC(b) protein (SEQ ID NO:2) to be producedinclude Bacillus thuringiensis and other Bacillus species such as B.subtilis or B. megaterium. It should be evident that genetically alteredor engineered microorganisms containing the cryIIIC(b) gene (SEQ IDNO:1) can also contain other toxin genes present in the samemicroorganism and that these genes could concurrently produceinsecticidal crystal proteins different from the CryIIIC(b) protein.

The Bacillus strains described in this disclosure may be cultured usingconventional growth media and standard fermentation techniques. The B.t.strains harboring the cryIIIC(b) gene (SEQ ID NO:1) may be fermented, asdescribed in the Examples, until the cultured B.t. cells reach the stageof their growth cycle when CryIIIC(b) crystal protein (SEQ ID NO:2) isformed. For sporogenous B.t. strains, fermentation is typicallycontinued through the sporulation stage when the CryIIIC(b) crystalprotein is formed along with spores. The B.t. fermentation culture isthen typically harvested by centrifugation, filtration or the like toseparate fermentation culture solids, containing the CryIIIC(b) crystalprotein, from the aqueous broth portion of the culture.

The B.t. strains exemplified in this disclosure are sporulatingvarieties (spore forming or sporogenous strains) but the cryIIIC(b) gene(SEQ ID NO:1) also has utility in asporogenous Bacillus strains, i.e.,strains that produced the crystal protein without production of spores.It should be understood that references to "fermentation cultures" ofB.t. strains (containing the cryIIIC(b) gene (SEQ ID NO:1)) in thisdisclosure are intended to cover sporulated B.t. cultures, i.e., B.t.cultures containing the CryIIIC(b) crystal protein and spores, andsporogenous Bacillus strains that have produced crystal protein duringthe vegetative stage, as well as asporogenous Bacillus strainscontaining the cryIIIC(b) gene (SEQ ID NO:1) in which the culture hasreached the growth stage where crystal protein is actually produced.

The separated fermentation solids are primarily CryIIIC(b) crystalprotein (SEQ ID NO:2) and B.t. spores, along with some cell debris, someintact cells, and residual fermentation medium solids. If desired, thecrystal protein may be separated from the other recovered solids viaconventional methods, e.g., sucrose density gradient fractionation.Highly purified CryIIIC(b) protein (SEQ ID NO:2) may be obtained bysolubilizing the recovered crystal protein and then precipitating theprotein from solution.

The CryIIIC(b) protein (SEQ ID NO:2), as noted earlier, is a potentinsecticidal compound against coleopteran insects, such as the Coloradopotato beetle. Japanese beetle larvae (white grubs), Mexican bean beetleand the like. The CryIIIC(b) protein (SEQ ID NO:2), in contrast to theCryIIIA and CryIIIB proteins, exhibits measurable insecticidal activityagainst Diabrotica insects, e.g., corn rootworms, which have beenrelatively unaffected by other coleopteran-toxic B.t. crystal proteins.The CryIIIC(b) protein (SEQ ID NO:2) may be utilized as the activeingredient in insecticidal formulations useful for the control ofcoleopteran insects such as those mentioned above. Such insecticidalformulations or compositions typically contain agriculturally acceptablecarriers or adjuvants in addition to the active ingredient and areprepared and used in a manner well known to those skilled in the art.

The CryIIIC(b) protein (SEQ ID NO:2) may be employed in insecticidalformulations in isolated or purified form, e.g., as the crystal proteinitself. Alternatively, the CryIIIC(b) protein (SEQ ID NO:2) may bepresent in the recovered fermentation solids, obtained from culturing ofa Bacillus strain, e.g., Bacillus thuringiensis, or other microorganismhost carrying the cryIIIC(b) gene (SEQ ID NO:1) and capable of producingthe CryIIIC(b) protein. Preferred Bacillus hosts include B.t. strainEG5144 and genetically improved B.t. strains derived from B.t. strainEG5144. The latter B.t. strains may be obtained via plasmid curingand/or conjugation techniques and contain the native cryIIIC(b)gene-containing plasmid from B.t. strain EG5144. Genetically engineeredor transformed B.t. strains or other host microorganisms containing arecombinant plasmid that expresses the cloned cryIIIC(b) gene (SEQ IDNO:1), obtained by recombinant DNA procedures, may also be used.

An example of such transformants is B.t. strain EG7237, which containsthe cloned cryIIIC(b) gene (SEQ ID NO:1) on a recombinant plasmid.

The recovered fermentation solids contain primarily the crystal proteinand (if a sporulating B.t. host is employed) spores; cell debris andresidual fermentation medium solids may also be present. The recoveredfermentation solids containing the CryIIIC(b) protein may be dried, ifdesired, prior to incorporation in the insecticidal formulation.

The formulations or compositions of this invention containing theinsecticidal CryIIIC(b) protein (SEQ ID NO:2) as the active componentare applied at an insecticidally effective amount which will varydepending on such factors as, for example, the specific coleopteraninsects to be controlled, the specific plant or crop to be treated andthe method of applying the insecticidally active compositions. Aninsecticidally effective amount of the insecticide formulation isemployed in the insect control method of this invention.

The insecticide compositions are made by formulating the insecticidallyactive component with the desired agriculturally acceptable carrier. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral) or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term "agriculturally acceptable carrier" covers all adjuvants,e.g., inert components, dispersants, surfactants, tackifiers, binders,etc. that are ordinarily used in insecticide formulation technology;these are well known to those skilled in insecticide formulation.

The formulations containing the CryIIIC(b) protein (SEQ ID NO:2) and oneor more solid or liquid adjuvants are prepared in known manners, e.g.,by homogeneously mixing, blending and/or grinding the insecticidallyactive CryIIIC(b) protein component with suitable adjuvants usingconventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran insect, typically onto the foliageof the plant or crop to be protected, by conventional methods,preferably by spraying. Other application techniques, e.g., dusting,sprinkling, soaking, soil injection, seed coating, seedling coating orspraying, or the like, are also feasible and may be required for insectsthat cause root or stalk infestation. These application procedures arewell known in the art.

The cryIIIC(b) gene (SEQ ID NO:1) or its functional equivalent,hereinafter sometimes referred to as the "toxin gene," can be introducedinto a wide variety of microorganism hosts. Expression of the cryIIIC(b)gene (SEQ ID NO:1) results in the production of insecticidal CryIIIC(b)crystal protein toxin (SEQ ID NO:2). Suitable hosts include B.t. andother species of Bacillus, such as B. subtilis or B. megaterium, forexample. Plant-colonizing or root-colonizing microorganisms may also beemployed as the host for the cryIIIC(b) gene (SEQ ID NO:1). Variousprocedures well known to those skilled in the art are available forintroducing the cryIIIC(b) gene (SEQ ID NO:1) into the microorganismhost under conditions which allow for stable maintenance and expressionof the gene in the resulting transformants.

The transformants, i.e., host microorganisms that harbor a cloned genein a recombinant plasmid, can be isolated in accordance withconventional methods, usually employing a selection technique, whichallows growth of only those host microorganisms that contain arecombinant plasmid. The transformants then can be tested forinsecticidal activity. Again, these techniques are standard procedures.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the gene into thehost, availability of expression systems, efficiency of expression,stability of the CryIIIC(b) insecticidal protein in the host, and thepresence of auxiliary genetic capabilities. The cellular host containingthe insecticidal cryIIIC(b) gene (SEQ ID NO:1) may be grown in anyconvenient nutrient medium, where expression of the cryIIIC(b) gene isobtained and CryIIIC(b) protein (SEQ ID NO:2) produced, typically tosporulation. The sporulated cells containing the crystal protein maythen be harvested in accordance with conventional methods, e.g.,centrifugation or filtration.

The cryIIIC(b) gene (SEQ ID NO:1) may also be incorporated into a plantwhich is capable of expressing the gene and producing CryIIIC(b) protein(SEQ ID NO:2), rendering the plant more resistant to insect attack.Genetic engineering of plants with the cryIIIC(b) gene (SEQ ID NO:1) maybe accomplished by introducing the desired DNA containing the gene intoplant tissues or cells, using DNA molecules of a variety of forms andorigins that are well known to those skilled in plant geneticengineering. An example of a technique for introducing DNA into planttissue is disclosed in European Patent Application Publication No. 0 289479, published Nov. 2, 1988, of Monsanto Company.

DNA containing the cryIIIC(b) gene (SEQ ID NO:1) or a modifiedcryIIIC(b) gene capable of producing the CryIIIC(b) protein (SEQ IDNO:2) may be delivered into the plant cells or tissues directly byinfectious plasmids, such as Ti, the plasmid from Agrobacteriumtumefaciens, viruses or microorganisms like A. tumefaciens, by the useof lysosomes or liposomes, by microinjection by mechanical methods andby other techniques familiar to those skilled in plant geneticengineering.

Variations may be made in the cryIIIC(b) gene nucleotide base sequence(SEQ ID NO:1), since the various amino acids forming the protein encodedby the gene usually may be determined by more than one codon, as is wellknown to those skilled in the art. Moreover, there may be somevariations or truncation in the coding regions of the cryIIIC(b)nucleotide base sequence which allow expression of the gene andproduction of functionally equivalent forms of the CryIIIC(b)insecticidal protein. These variations which can be determined withoutundue experimentation by those of ordinary skill in the art withreference to the present specification are to be considered within thescope of the appended claims, since they are fully equivalent to thespecifically claimed subject matter.

The present invention will now be described in more detail withreference to the following specific, non-limiting examples. The examplesrelate to work which was actually done based on techniques generallyknown in the art and using commercially available equipment.

The novel B.t. strain EG5144 was isolated following the proceduredescribed in Example 1. The procedures described in Example 1 were alsoused to isolate the novel B.t. strain EG5145.

EXAMPLE 1 Isolation of B.t. Strains EG5144 and EG5145

Crop dust samples were obtained from various sources throughout the U.S.and abroad, typically grain storage facilities. The crop dust sampleswere treated by suspending the crop dust in an aqueous buffer andheating the suspension at 60° C. for 30 min. to enrich for heatresistant spore forming Bacillus-type bacteria such as B.t. The treateddust suspensions were diluted in aqueous buffer, and the dilutions werespread on agar plates to allow each individual bacterium from the cropdust to grow into a colony on the surface of the agar plate. Aftergrowth, a portion of each colony was transferred from the agar plate toa nitrocellulose filter. The filter was treated with NaOH to lyse thecolonies and to fix the DNA from each colony onto the filter.

A modified treatment procedure was developed for use with B.t. coloniesutilized in the colony hybridization procedure, since standardtechniques applicable to E. coli were found to be unworkable with B.t.In the treatment described above, special conditions were required toassure that the B.t. colonies were in a vegetative state of growth,making them susceptible to lysis with NaOH. Accordingly, after a portionof each colony was transferred to the nitrocellulose filter, the filterwas placed colony side up on an agar medium containing 0.5% (w/v)glucose. The transferred colonies were then allowed to grow on theagar-glucose medium for 5 hours at 30° C. Use of 0.5% glucose in theagar medium and the 5-hour, 30° C. growth cycle were critical forassuring that the B.t. colonies were in a vegetative state and thussusceptible to lysis.

A cloned coleopteran toxin gene was used as a specific probe to findother novel and rare coleopteran-toxic strains of B.t. from crop dustsamples. A 2.9 kb HindIII DNA restriction fragment containing thecryIIIA gene, formerly known as the cryC gene of B.t. strain EG2158,described in Donoran et al., Mol. Gen. Genet., 214, pp.365-372 (1988),was used as a probe in colony hybridization procedures.

The 2.9 kb HindIII cryIIIA DNA fragment, containing the entire cryIIIAgene, was radioactively labeled with [alpha-P³² ]-dATP and Klenowenzyme, by standard methods. The nitrocellulose filters containing theDNA from each lysed colony were incubated at 65° C. for 16 hours in abuffered solution that contained the radioactively labeled 2.9 kbHindIII cryIIIA DNA probe to hybridize the DNA from the colonies withthe DNA from the radioactively labeled cryIIIA probe. The 65° C.hybridization temperature was used to assure that the cryIIIA DNA probewould hybridize only to DNA from colonies that contained a gene that wassimilar to the cryIIIA DNA probe.

The 2.9 kb cryIIIA probe hybridized to many B.t. colonies from varioussamples of crop dust. Examination of these colonies revealed,unexpectedly, that they did not contain any cryIII-type genes. Thesecolonies did contain cryI-type genes. The cryI-type genes encodelepidopteran-toxic, coleopteran-nontoxic crystal proteins with molecularmasses of approximately 130 kDa. Computer-assisted comparisons of thesequence of the cryIIIA gene with the sequence of several cryI-typegenes revealed that the 3'-end of the cryIIIA gene was partiallyhomologous with portion of the cryI-type genes. This finding supportedthe belief that the 3'-end of the cryIIIA gene was causing the 2.9 kbcryIIIA probe to hybridize to B.t. colonies containing cryI-type genes.

To correct this problem, the 2.9 kb HindIII cryIIIA probe was digestedwith the enzyme XbaI and a 2.0 kb HindIII-XbaI fragment was purifiedthat contained the cryIIIA gene minus its 3'-end. The 2.0 kbHindIII-XbaI fragment contains the 3'-truncated cryIIIA gene. When the2.0 kb fragment was used in repeated colony hybridization experiments,it did not hybridize to cryI gene-containing B.t. colonies.

Approximately 48,000 Bacillus-type colonies from crop dust samples fromvarious locations were probed with the radioactively labeled 2.0 kbHindIII-XbaI cryIIIA probe. Only one novel B.t. strain from an Illinoiscrop dust sample was discovered that specifically hybridized to thecryIIIA probe. That novel strain was designated B.t. strain EG2838,which has been deposited with the NRRL under Accession No. NRRL B-18603.

Subsequently, approximately 50,000 additional Bacillus-type coloniesfrom crop dust samples were also screened with the radioactively labeled2.0 kb HindIII-XbaI cryIIIA probe, but without success in identifyingany other strains containing novel cryIII-type genes.

B.t. strain EG2838 was found to be insecticidally active againstcoleopteran insects, notably, the Colorado potato beetle. B.t. strainEG2838 did not have substantial insecticidal activity with respect tothe southern corn rootworm. A gene, designated the cryIIIB gene, wasisolated from B.t. strain EG2838, and its nucleotide base sequencedetermined. The cryIIIB gene encoded a crystal protein, designated theCryIIIB protein, containing 651 amino acids having a deduced size of74,237 Daltons. The size of the prior art CryIIIA protein had previouslybeen deduced to be 73,116 Daltons (644 amino acids). The cryIIIB gene is75% homologous with the cryIIIA gene, and the CryIIIB protein is 68%homologous with the CryIIIA protein.

Thousands of Bacillus-type colonies from numerous crop dust samples fromvarious locations from around the world were screened with a cryIIIBprobe obtained from B.t. strain EG2838. The cryIIIB probe wasradioactively labeled using the procedure set forth above with respectto the radioactively labeled cryIIIA probe. The radioactively labeledcryIIIB probe consisted of a 2.4 kb SspI restriction fragment of DNAfrom B.t. strain EG2838. The fragment contains the complete proteincoding region for the coleopteran toxin cryIIIB gene of B.t. strainEG2838. Ultimately, the B.t. strains of the present invention,designated B.t. strains EG5144 and EG5145, were isolated from a cropdust sample via B.t. colonies that specifically hybridized to thecryIIIB probe.

To characterize B.t. strain EG5144, several studies were conducted. Oneseries of studies was performed to characterize its flagellar serotype.Additional studies were conducted to determine the sizes of the nativeplasmids in B.t. strain EG5144 and to ascertain which plasmids containedgenes that encoded coleopteran-active insecticidal crystal proteins. DNAblot analysis was thereafter performed using size fractionated total DNArestriction fragments from B.t. strain EG5144, compared withsimilarly-processed total DNA from other B.t. strains containingcryIII-type toxin genes, to demonstrate that B.t. strain EG5144 containsa unique coleopteran-active toxin gene. In addition, B.t. strain EG5144was evaluated further by characterizing the crystal proteins it producesand by measuring the insecticidal activity associated with B.t. strainEG5144 and its crystal proteins. Examples 2 through 7 are directed tothe procedures for characterizing B.t. strain EG5144 and its uniqueCryIII-type gene, and Examples 8 through 11 are directed to theinsecticidal activity of B.t. strain EG5144 and of B.t. strain EG7237,containing the cryIIIC(b) gene (SEQ ID NO:1) of this invention.

EXAMPLE 2 Evaluation of the Flagellar Serotype of B.t. Strain EG5144

Flagellar serotyping studies were carried out with B.t. strain EG5144,using an antibody mediated cell agglutinization assay (Craigie et al.,J.Immunol., 21, pp.417-511 (1936)). Flagellar antibody reagents wereprepared using purified flagella from B.t. var. kurstaki, morrisoni andtolworthi type-strains and from the novel coleopteran-active B.t. strainEG4961.

The study included formalin-fixed vegetative cells of B.t. strain EG5144and of cells of other coleopteran-active B.t. strains and of severalcommon B.t. type-strains, each of which were scored for flagellarantibody mediated cell agglutinization.

The other coleopteran-active B.t. strains included B.t. var.tenebrionis, B.t. var. san diego, B.t. strain EG2158 (all containing thecryIIIA gene); B.t. strain EG2838 (containing the cryIIIB gene); andB.t. strain EG4961 (containing a novel coleopteran toxin-encoding genedesignated as the cryIIIC(a) gene).

The B.t. flagellar type-strains were B.t. var. kurstaki (HD-1, serotype3ab), B.t. var. morrisoni (HD-12, serotype 8ab) and B.t. var. tolworthi(HD-13, serotype 9).

Results of this study are shown in Table 1; "+" indicates that across-reaction occurred and "-" indicates that no cross-reactionoccurred.

                  TABLE 1                                                         ______________________________________                                                   Flagellar Antibody Reagent                                         Cells        kurstaki morrisoni                                                                              tolworthi                                                                            EG4961                                  ______________________________________                                        B.t. strain EG5144                                                                         -        -        -      -                                       B.t. var. tenebrionis                                                                      -        +        -      -                                       B.t. var. san diego                                                                        -        +        -      -                                       B.t. strain EG2158                                                                         -        +        -      -                                       B.t. strain EG2838                                                                         -        -        +      -                                       B.t. strain EG4961                                                                         -        -        -      +                                       Other B.t. flagellar                                                          type-strains:                                                                 B.t. var. kurstaki                                                                         +        -        -      -                                       (HD-1)                                                                        B.t. var. morrisoni                                                                        -        +        -      -                                       (HD-12)                                                                       B.t. var. tolworthi                                                                        -        -        +      -                                       (HD-13)                                                                       ______________________________________                                    

The results in Table 1 show that cells of B.t. strain EG5144 gave anegative reaction with B.t. type-strain kurstaki, morrisoni andtolworthi flagella antibody reagents. B.t. strain EG5144 cells also gavea negative reaction with flagellar reagent from B.t. strain EG4961, anovel coleopteran-active strain that has been discovered to exhibitDiabrotica toxicity.

These results indicate that B.t. strain EG5144 is not a kurstaki,morrisoni or tolworthi-type B.t. strain. Furthermore, the flagellarserotype of B.t. strain EG5144, which is yet not known, is apparentlydifferent from that of B.t. strain EG4961, which has been serotyped asserovar kumamotoensis (serotype 18). Both B.t. strain EG5144 and B.t.strain EG4961 appear to have flagellar serotypes that are different fromthose of other coleopteran-toxic B.t. strains reported in theliterature.

EXAMPLE 3 Size Fractionation and cryIIIB Probing of Native Plasmids ofEG5144

B.t. strains may be characterized by fractionating their plasmidsaccording to size by the well-known procedure of agarose gelelectrophoresis. This procedure involves lysing B.t. cells with lysozymeand SDS, electrophoresing plasmids from the lysate through an agarosegel and staining the gel with ethidium bromide to visualize theplasmids. Larger plasmids, which move more slowly through the gel,appear at the top of the gel and smaller plasmids appear toward thebottom of the gel.

The agarose gel in FIG. 2 shows that B.t. strain EG5144 contains nativeplasmids of approximately 145, 92, 12, 10 and 5.5 MDa, as indicated bythe white horizontal bands. Plasmid sizes were estimated by comparisonto plasmids of known sizes (not shown). Although not shown on FIG. 2,B.t. strain EG5145 contains native plasmids of approximately 145, 92, 12and 5.5 MDa. The cryptic 10 MDa plasmid found in B.t. strain EG5144 isnot present in B.t. strain EG5145.

FIG. 2 further shows that the coleopteran-toxic B.t. strain EG4961contains native plasmids of about 150, 95, 70, 50, 5 and 1.5 MDa andthat the coleopteran-toxic B.t. strain EG2838 contains native plasmidsof about 100, 90 and 37 MDa. FIG. 2 also shows that thecoleopteran-toxic B.t. strain EG2158 contains native plasmids of about150, 105, 88, 72, and 35 MDa. Some of the plasmids, such as the 150 and1.5 MDa plasmids of B.t. strain EG4961 and the 150 MDa plasmid of B.t.strain EG2158, may not be visible in the photograph, although they arevisible in the actual gel. FIG. 2 demonstrates that the sizes of thenative plasmids of B.t. strain EG5144 are different from the sizes ofthe native plasmids of B.t. strains EG2158, EG2838 and EG4961. B.t.strain EG5144 is therefore distinct from the other coleopteran-toxicB.t. strains EG2158, EG2838 and EG4961, based on these plasmid arraystudies and on the serotyping studies described in Example 2. Likewise,B.t. strain EG5145 appears distinct from the coleopteran-toxic B.t.strains noted above based on plasmid array studies.

The plasmids shown in FIG. 2 were transferred by blotting from theagarose gel to a nitrocellulose filter using the blot techniques ofSouthern, J.Molec.Biol., 98, pp.503-517 (1975), and the filter washybridized as described above with the radioactively labeled 2.4 kbcryIIIB DNA probe. After hybridization, the filter was exposed to X-rayfilm. Examination of the X-ray film confirmed that the cryIIIB probespecifically hybridized to the 92 MDa plasmid of B.t. strain EG5144.This result demonstrates that the 92 MDa plasmid of B.t. strain EG5144contains a DNA sequence that is at least partly homologous to thecryIIIB gene and confirms that the 92 MDa plasmid contains a cryIII-typegene. The X-ray film also showed that the cryIIIB probe hybridized, asexpected, to the 95 MDa plasmid of B.t. strain EG4961 and to the 100 MDaplasmid of B.t. strain EG2838, and to the 88 MDa plasmid of B.t. strainEG2158. The 88 MDa plasmid of B.t. strain EG2158 has been previouslyshown to contain the coleopteran-toxin cryIIIA gene (see Donovan et al.,Mol.Gen.Genet., 214, pp.365-372 (1988)). The inventors have previouslydetermined that the 100 MDa plasmid of B.t. strain EG2838 contains thecoleopteran toxin cryIIIB gene and that the 95 MDa plasmid of B.t.strain EG4961 contains the novel coleopteran toxin cryIIIC(a) gene.

EXAMPLE 4 Blot Analysis of DNA from B.t. Strains EG5144 and EG5145

Both chromosomal and plasmid DNA (total DNA) from B.t. strain EG5144were extracted and digested with separate restriction enzymes, SspI,HindIII and EcoRI. The digested DNA was size fractionated byelectrophoresis through an agarose gel, and the fragments were thenvisualized by staining with ethidium bromide. For comparison, total DNAfrom the coleopteran-toxic B.t. strains EG2158, EG2838 and EG4961 wasprocessed in an identical manner. Examination of the resultant stainedagarose gel showed that restriction digestions of total DNA from theseB.t. strains with each of SspI, HindIII and EcoRI yield hundreds of DNAfragments of various sizes.

The size fractionated DNA restriction fragments were transferred byblotting from the agarose gel to a nitrocellulose filter and were thenprobed with a cryIII-type DNA hybridization probe. The filter washybridized at 65° C. in a buffered aqueous solution containing aradioactively labeled 2.4 kb cryIIIB DNA probe. After hybridization, thefilter was exposed to X-ray film to make an autoradiogram. FIG. 3 is aphotograph of the autoradiogram where the numbers to the left indicatethe size, in kb, of the DNA fragments of B.t. strain EG5144 thathybridized to the cryIIIB probe. These sizes were determined bycomparison with the lane labeled "stnd" which contained phage lambda DNAdigested with HindIII and radioactively labelled as size markers. Lanesin FIG. 3 marked EG2158, EG5144, EG2838 and EG4961 contain sizefractionated DNA fragments from these respective B.t. strains, obtainedby digestion with the restriction enzyme designated above the individuallanes.

In the lanes for each B.t strain in FIG. 3, the dark bands represent DNArestriction fragments that hybridized with the cryIIIB probe. Visualinspection of FIG. 3 shows that the sizes of the cryIIIB-hybridizingrestriction fragments of B.t. strain EG5144 are distinctly differentfrom the sizes of the cryIIIB-hybridizing fragments of B.t. strainsEG2158, EG2838 and EG4961.

In particular, the size of the cryIIIB-hybridizing SspI restrictionfragment for B.t. strain EG5144 is 3.4 kb, and this is unlike thecorresponding SspI restriction fragments for the other three B.t.strains: 2.8 kb for B.t. strain EG2158; 2.4 kb for B.t. strain EG2838;and 4.5 and 6.0 kb for B.t. strain EG4961. Similar differences areapparent for the DNA restriction fragments obtained using HindIII andEcoRI.

These restriction pattern results suggest that B.t. strain EG5144contains a cryIII-type gene that is different from the cryIIIA, cryIIIBand cryIIIC(a) genes of B.t. strains EG2158, EG2838 and EG4961,respectively. The cryIII-type gene of B.t. strain EG5144 has beendesignated cryIIIC(b) (SEQ ID NO:1) by the inventors.

Total DNA from B.t. strain EG5144 and B.t. strain EG5145 was extractedand digested with six separate restriction enzymes (HindIII, EcoRI,AccI, DraI, SspI, XbaI), and size fractionated by electrophoresis on anagarose gel. The size fractionated DNA restriction fragments were thentransferred by blotting to a nitrocellulose filter and were then probedwith a cryIII-type DNA hybridization probe, specifically a probecontaining cryIIIA. After hybridization, the filter was exposed to X-rayfilm to make an autoradiogram. The restriction pattern results wereidentical for the two B.t. strains evaluated, EG5144 and EG5145, whichsuggests that the two strains contain the same cryIII-type gene.

EXAMPLE 5 Characterization of Crystal Proteins of B.t. Strain EG5144

B.t. strain EG5144 was grown in DSMG sporulation medium at roomtemperature (about 21°-25° C.) until sporulation and cell lysis hadoccurred (4 to 5 days growth). The DSMG medium is 0.4% (w/v) Difconutrient broth, 25 mM K₂ HPO₄, 25 mM KH₂ PO₄, 0.5 mM Ca(NO₃)₂, 0.5 mMMgSO₄, 10 μM FeSO₄, 10 μM MnCl₂ and 0.5% (w/v) glucose. The sporulatedculture of B.t. strain EG5144 was observed microscopically to containfree floating, irregularly shaped crystals in addition to B.t. spores.Experience has shown that B.t. crystals are usually composed of proteinsthat may be toxic to specific insects. The appearance of the crystals ofB.t. strain EG5144 differed from the flat, rectangular (or rhomboidal)crystals of B.t. strain EG2158, but partially resembled some of theirregularly shaped crystals of B.t. strains EG2838 and EG4961.

Spores, crystals and residual lysed cell debris from the sporulatedculture of B.t. strain EG5144 were harvested by centrifugation. Therecovered solids were washed once with aqueous 1N NaCl and twice withTETX (containing 10 mM Tris HCl pH 7.5, 1 mM EDTA and 0,005% (w/v)Triton® X-100) and suspended in TETX at a concentration of 50 mg/ml. Thewashed crystals were specifically solubilized from 250 μg centrifugedfermentation culture solids (containing crystals, spores and some celldebris) by heating the solids mixture in a solubilization buffer (0.14MTris pH 6.8, 2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v)glycerol and 0.1% (v/v) bromophenol blue) at 100° C. for 5 minutes. Thesolubilized crystal proteins were size fractionated by SDS-PAGE. Aftersize fractionation, the proteins were visualized by staining withCoomassie dye. Cultures of B.t. strains EG4961, EG2158 and EG2838 wereprocessed in an identical manner for purposes of comparison.

FIG. 4 shows the results of this protein size fractionation analysiswhere the numbers to the left indicate the size, in kDa, of the crystalproteins synthesized by B.t. strain EG5144. As shown in lane 1, a majorprotein of approximately 70 kDa and a minor protein of approximately 30kDa were solubilized from centrifuged fermentation solids containingB.t. strain EG5144 spores and crystals. The approximately 70 kDa proteinof B.t. strain EG5144 appears similar in size to the approximately 70kDa coleopteran-toxic crystal proteins of B.t. strains EG4961 (lane 2),EG2158 (lane 3) and to the approximately 74 kDa coleopteran-toxiccrystal protein of B.t. strain EG2838 (lane 4).

Previous work by the inventors has shown that the coleopteran-toxiccrystal proteins of B.t. strains EG4961, EG2158 and EG2838 are eachdifferent. The CryIIIC(a) protein of B.t. strain EG4961 is coded by thecryIIIC(a) gene and has a deduced size of 74,393 Da. The CryIIIA proteinof B.t. strain EG2158 is coded by the cryIIIA gene and has a deducedsize of 73,116 Da. The CryIIIB protein of B.t. strain EG2838 is coded bythe cryIIIB gene and has a deduced size of 74,237 Da. As described inExample 6, the coleopteran-toxic crystal protein of B.t. strain EG5144produced by the novel cryIIIC(b) gene (SEQ ID NO:1) is clearly differentfrom the CryIIIA, CryIIIB and CryIIIC(a) proteins.

The minor crystal protein of approximately 30 kDa that is produced byB.t. strain EG5144 is roughly similar in size to small crystal proteinsproduced by B.t. strains EG4961, EG2158 and EG2838. The approximately 30kDa minor proteins of B.t. strains EG2158, EG2838 and EG4961 appear tobe related to each other and none has been found to exhibit measurableinsecticidal activity towards coleopteran insects. There is no reason tobelieve that the approximately 30 kDa protein of B.t. strain EG5144possesses insecticidal activity against coleopteran insects.

Following the procedure of Example 4, further DNA blot analysis revealedthat the 2.4 kb cryIIIB DNA probe specifically hybridized to a single7.0 kb EcoRI-XbaI restriction fragment of B.t. strain EG5144 DNA. Thisresult suggested that the 7.0 kb fragment contained the completecryIIIC(b) gene.

The 7.0 kb EcoRI-XbaI fragment of B.t. strain EG5144 was isolated andstudies were conducted on the 7.0 kb EcoRI-XbaI restriction fragment toconfirm that the fragment contained a cryIII-type gene, in particular,the cryIIIC(b) gene. The procedures set forth in Example 6 describe thedetermination of the nucleotide base sequence of the cryIIIC(b) gene(SEQ ID NO:1).

EXAMPLE 6 Cloning and Sequencing of the cryIIIC(b) Gene of B.t. StrainEG5144

In order to isolate the 7.0 kb EcoRI-XbaI fragment described in theprevious Example, a plasmid library of B.t. strain EG5144 wasconstructed by ligating size-selected DNA EcoRI-XbaI restrictionfragments from B.t. strain EG5144 into the well-known E. coli vectorpUC18. This procedure involved first obtaining total DNA from B.t.strain EG5144 by cell lysis followed by DNA spooling, then doubledigesting the total DNA with both EcoRI and XbaI restriction enzymes,electrophoresing the digested DNA through an agarose gel, excising a gelslice containing 4-10 kb size selected fragments of DNA, andelectroeluting the size selected EcoRI-XbaI restriction fragments fromthe agarose gel slice. These fragments were mixed with the E. coliplasmid vector pUC18, which had also been digested with EcoRI and XbaI.The pUC18 vector carries the gene for ampicillin resistance (Amp^(r))and the vector replicates in E. coli. T4 DNA ligase and ATP were addedto the mixture of size-selected restriction fragments of DNA from B.tstrain EG5144 and of digested pUC18 vector to allow the pUC18 vector toligate with the B.t. strain EG5144 restriction fragments.

The plasmid library was then transformed into E. coli cells, a hostorganism lacking the gene of interest, as follows. After ligation, theDNA mixture was incubated with an ampicillin sensitive E. coli hoststrain, E. coli strain DH5α, that had been treated with CaCl₂ to allowthe cells to take up the DNA. E. coli, specifically strain DH5α, wasused as the host strain because these cells are easily transformed withrecombinant plasmids and because E. coli strain DH5αdoes not naturallycontain genes for B.t. crystal proteins. Since pUC18 confers resistanceto ampicillin, all host cells acquiring a recombinant plasmid wouldbecome ampicillin resistant. After exposure to the recombinant plasmids,the E. coli host cells were spread on agar medium that containedampicillin. After incubation overnight at a temperature of 37° C.,several thousand E. coli colonies grew on the ampicillin-containing agarfrom those cells which harbored a recombinant plasmid. These E. colicolonies were then blotted onto nitrocellulose filters for subsequentprobing.

The radioactively labeled 2.4 kb cryIIIB gene was then used as a DNAprobe under conditions that permitted the probe to bind specifically tothose transformed host colonies that contained the 7.0 kb EcoRI-XbaIfragment of DNA from B.t. strain EG5144. Several E. coli coloniesspecifically hybridized to the 2.4 kb cryIIIB probe. OnecryIIIB-hybridizing colony, designated E. coli strain EG7236, wasstudied further. E. coli strain EG7236 contained a recombinant plasmid,designated pEG271, which consisted of pUC18 plus the inserted EcoRI-XbaIrestriction fragment of DNA from B.t. strain EG5144 of approximately 7.0kb. The cryIIIB probe specifically hybridized to the 7.0 kb DNA fragmentinsert in pEG271. A restriction map of pEG271 is shown in FIG. 5.

The 7.0 kb fragment of pEG271 contained HindIII fragments of 2.4 kb and3.8 kb, and a BamHI-XbaI fragment of 4.0 kb that specifically hybridizedwith the cryIIIB probe. The 2.4 kb HindIII fragment was subcloned intothe DNA sequencing vector M13mp18. The 4.0 kb BamHI-XbaI fragment wassubcloned into the DNA sequencing vectors M13mp18 and M13mp19.

The nucleotide base sequence of a substantial part of each subcloned DNAfragment was determined using the standard Sanger dideoxy method. Foreach subcloned fragment, both DNA strands were sequenced by usingsequence-specific 17-mer olignucleotide primers to initiate the DNAsequencing reactions. Sequencing revealed that the 7.0 kb fragmentcontained an open reading frame and, in particular, a new cryIII-typegene. This new gene, designated cryIIIC(b) (SEQ ID NO:1), issignificantly different from the cryIIIA gene. As indicated below, thecryIIIC(b) gene is also clearly distinct from the cryIIIB gene.

The DNA sequence of the cryIIIC(b) gene (SEQ ID NO:1) and the deducedamino acid sequence of the CryIIIC(b) protein (SEQ ID NO:2) encoded bythe cryIIIC(b) gene are shown in FIG. 1. The protein coding portion ofthe cryIIIC(b) gene (SEQ ID NO:1) is defined by the nucleotides startingat position 144 and ending at position 2099. The probable ribosomebinding site is indicated as "RBS" in FIG. 1A. The size of theCryIIIC(b) protein (SEQ ID NO:2) encoded by the cryIIIC(b) gene, asdeduced from the open reading frame of the cryIIIC(b) gene (SEQ IDNO:1), is 74,265 Da (652 amino acids). It should be noted that theapparent size of the CryIIIC(b) protein, as determined from SDS-PAGE, isapproximately 70 kDa. Therefore, the CryIIIC(b) protein (SEQ ID NO:2)will be referred to in this specification as being approximately 70 kDain size.

The size of the prior art CryIIIA protein has previously been deduced tobe 73,116 Da (644 amino acids). The size of the CryIIIB protein haspreviously been determined to be 74,237 Da (651 amino acids).

DNA sequencing revealed the presence of a HindIII restriction sitewithin the cryIIIC(b) gene and a SspI restriction site downstream of thecryIIIC(b) gene (See FIGS. 1B and 1C respectively). Knowledge of thelocations of these restriction sites permitted the precise determinationof the location and orientation of the cryIIIC(b) gene within the 7.0 kbfragment as indicated by the arrow in FIG. 5.

The computer program of Korn and Queen (L. J. Korn and C. Queen,"Analysis of Biological Sequences on Small Computers," DNA, 3, pp.421-436 (1984)) was used to compare the sequences of the cryIIIC(b) gene(SEQ ID NO:1) to the cryIIIB and cryIIIA genes and to compare thededuced amino acid sequences of their respective CryIIIC(b), CryIIIB andCryIIIA proteins.

The nucleotide base sequence of the cryIIIC(b) gene (SEQ ID NO:1) was96% positionally identical with the nucleotide base sequence of thecryIIIB gene and only 76% positionally identical with the nucleotidebase sequence of the cryIIIA gene. Thus, although the cryIIIC(b) gene(SEQ ID NO:1) is related to the cryIIIB and cryIIIA genes, it is clearthat the cryIIIC(b) gene is distinct from the cryIIIB gene andsubstantially different from the cryIIIA gene.

The deduced amino acid sequence of the CryIIIC(b) protein (SEQ ID NO:2)was found to be 95% positionally identical to the deduced amino acidsequence of the CryIIIB protein, but only 68% positionally identical tothe deduced amino acid sequence of the CryIIIA protein. Thesedifferences, together with the differences in insecticidal activity asset forth below, clearly show that the CryIIIC(b) protein encoded by thecryIIIC(b) gene (SEQ ID NO:1) is a different protein from the CryIIIBprotein or the CryIIIA protein.

Moreover, while not wishing to be bound by any theory, based on acomparison of the amino acid sequences of the CryIIIC(b) protein (SEQ IDNO:2) with other CryIII-type proteins known to the inventors, it isbelieved that the following amino acid residues may be of significancefor the enhanced corn rootworm toxicity of the CryIIIC(b) protein, wherethe numbers following the accepted abbreviations for the amino acidsindicate the position of the amino acid in the sequence illustrated inFIG. 1 and identified in SEQ ID NO:2: His9, His231, Gln339, Ser352,Asn446, His449, Val 450, Gly451, Ile600 and Thr624. These amino acidresidues were selected as being of probable significance for the cornrootworm toxicity of the CryIIIC(b) protein (SEQ ID NO:2) because, afterstudying the amino acid sequences of several other CryIII proteins, theamino acids at the indicated positions fairly consistently showeddifferent amino acids than those indicated for the CryIIIC(b) protein.

Based on the same studies, it is also believed that site directedmutagenesis of the cryIIIC(b) gene (SEQ ID NO:1) may result in improvedor enhanced corn rootworm toxicity for the resultant protein where oneor more of the following amino acid modifications are effected: Pro21 toGly; Asp97 to Asn; Val 289 to Ile; Ser352 to Phe; 417Ile to Val; Phe419to Leu; Gly45 to Ser; Ile590 to Leu; Ile600 to Lys; Thr624 to Lys.

As is well understood in the art, other changes in the cryIIIC(b) gene(SEQ ID NO:1) may be made, via site directed mutagenesis or genetruncation or the like, that could yield a toxic protein which possessesessentially similar insecticidal activity (to corn rootworm and othercoleopteran insects) as that exhibited by the CryIIIC(b) protein (SEQ IDNO:2). Modifications to the cryIIIC(b) gene (SEQ ID NO:1) and CryIIIC(b)protein (SEQ ID NO:2) such as described above are intended to be withinthe scope of the claimed invention.

EXAMPLE 7 Expression of the Cloned cryIIIC(b) Gene

Studies were conducted to determine the production of the CryIIIC(b)protein (SEQ ID NO:2) by the cryIIIC(b) gene (SEQ ID NO:1).

Table 2 summarizes the relevant characteristics of the B.t. and E. colistrains and plasmids used during these procedures. A plus (⁺) indicatesthe presence of the designated element, activity or function and a minus(⁻) indicates the absence of the same. The designations ^(s) and ^(r)indicate sensitivity and resistance, respectively, to the antibioticwith which each is used. The abbreviations used in the Table have thefollowing meanings: Amp (ampicillin); Cm (chloramphenicol); Cry(crystalliferous); Tc (tetracycline).

                  TABLE 2                                                         ______________________________________                                        Strains and Plasmids                                                          Strains   Relevant characteristics                                            ______________________________________                                        B. thuringiensis                                                              HD73-26   Cry.sup.-, Cm.sup.s                                                 EG7237    RD73-26 harboring pEG272 (cryIIIC(b).sup.+)                         EG5144    CryIIIC(b).sup.+                                                    DH5a      Cry.sup.-, Amps                                                     GM2163    Cry.sup.-, Amps                                                     EG7236    DH5a harboring pEG271 (CryIIIC(b).sup.+)                            Plasmids                                                                      pUC18     Amp.sup.r, Cry.sup.-, E. coli vector                                pNN101    Cm.sup.r, Tc.sup.r, Cry.sup.-, Bacillus vector                      pEG271    Amp.sup.r, CryIIIC(b).sup.+  E. coli                                          recombinant plasmid consisting of                                             the 7.0 kb EcoRI-XbaI cryII.TC(b).sup.+                                       fragment of B.t. strain EG5144                                                ligated into the EcoRI-XbaI sites                                             of pUC18                                                            pEG272    Tc.sup.r, Cm.sup.r, cryllIC(b).sup.+  Bacillus-E.                             coli recombinant plasmid                                                      consisting of the Bacillus vector                                             pNN101 ligated into the SphI site                                             of pEG271.                                                          ______________________________________                                    

E. coli cells harboring plasmid pEG271 described in Example 6 wereanalyzed and found not to produce detectable amounts of the 70 kDaCryIIIC(b) crystal protein.

Experience has shown that cloned B.t. crystal genes are poorly expressedin E. coli and highly expressed in B.t. from their respective nativepromoter sequences. Recombinant plasmid pEG271, constructed as set forthin Example 6 and shown in FIG. 5, will replicate in E. coli, but willnot replicate in B.t. To achieve a high level of expression of thecloned cryIIIC(b) gene, the Bacillus vector pNN101 (Tc^(r) Cm^(r) Cry⁻)that is capable of replicating in B.t. was ligated into the SphI site ofpEG271. The resultant plasmid was designated pEG272. Details of theconstruction of plasmid pEG272 and its subsequent use to transform B.t.are described below.

The isolated plasmid pEG271 DNA was digested with SphI and was thenmixed with the Bacillus vector pNN101 that had also been digested withSphI. T4 DNA ligase and ATP were added to the mixture to allow pEG271 toligate into the SphI site of the pNN101 vector.

After ligation, the DNA mixture was added to a suspension of E. colistrain DH5α cells that had been treated with calcium chloride to permitthe cells to take up plasmid DNA. After exposure to the recombinantplasmids, the E. coli host cells were spread on an agar mediumcontaining tetracycline. Only cells that had taken up a plasmidconsisting of pEG271 ligated into the SphI site of pNN101 would grow onthe tetracycline agar medium whereas cells that had not absorbed theplasmid would not grow.

Plasmid was isolated from one tetracycline resistant colony, digestedwith SphI, and electrophoresed through an agarose gel. The plasmidconsisted of two SphI DNA fragments of 5.8 kb and 9 kb corresponding toplasmids pNN101 and pEG271, respectively. This plasmid was designatedpEG272. A restriction map of pEG272 is shown in FIG. 6. Plasmid pEG272was then used to transform cells of E. coli strain GM2163 made competentby the calcium chloride procedure described earlier in Example 6. E.coli strain GM2163 is a crystal negative (Cry⁻) and ampicillin sensitive(Amp^(s)) strain, constructed by the procedures of M. G. Marinus et al.in Mol.Gen.Genet., 192, pp.288-289 (1983).

Plasmid pEG272 was then isolated from the transformed E. coli strainGM2163, using the procedures described above. The isolated plasmidpEG272 was next transformed by electroporation into B.t. strain HD73-26.Cells of B.t. strain HD73-26 are crystal-negative (Cry⁻) andchloramphenicol sensitive (Cm^(s)). Using a BioRad Gene Pulser™apparatus to carry out the electroporation, cells of B.t. strain HD73-26in suspension were induced to take up pEG272 which was also added to themixture.

After electroporation, the transformed B.t. cells were spread onto anagar medium containing 5 μg chloramphenicol and were incubated about16-18 hours at 30° C. Cells that had taken up plasmid pEG272 would growinto colonies on the chloramphenicol agar medium whereas cells that hadnot absorbed the plasmid would not grow. One Cm^(r) colony, designatedB.t. strain EG7237, contained a plasmid whose restriction patternappeared identical to that of pEG272.

Cells of B.t. strain EG7237 were grown in a sporulation mediumcontaining chloramphenicol (3 μg/ml) at 22°-25° C. until sporulation andcell lysis had occurred (4-5 days). Microscopic examination revealedthat the sporulated culture of B.t. strain EG7237 contained spores andsmall free floating irregularly shaped crystals. These crystalsresembled the small, irregularly-shaped crystals observed with asporulated culture of B.t. strain EG5144 that had been prepared in asimilar manner.

Spores, crystals and cell debris from the sporulated fermentationculture of B.t. strain EG7237 were harvested by centrifugation. Thecentrifuge pellet was washed once with 1N aqueous NaCl and twice withTETX (10 mM Tris.HCl pH 7.5, 1 mM EDTA, 0.005% (w/v) Triton® X-100), andthe pellet suspended in TETX at a concentration of 50 mg pellet/ml TETX.

The crystals in the centrifuge pellet suspension were solubilized byheating a portion of the centrifuge suspension (containing 250 μg pelletsolids) in solubilization buffer (0.14 M Tris pH 6.8, 2% (w/v) SDS, 5%(v/v) 2-mercaptoethanol, 10% (v/v) glycerol and 0.1% (w/v) bromophenolblue) at 100° C. for 5 minutes. After crystal solubilization hadoccurred, the mixture was applied to an SDS-polyacryamide gel and thesolubilized proteins in the mixture were size fractionated byelectrophoresis. After size fractionization, the proteins werevisualized by staining with Coomassie dye. A photograph of the Coomassiestained gel is shown in FIG. 7.

Lane 3 of the gel in FIG. 7 shows that B.t. strain EG7237 produced amajor protein of approximately 70 kDa and a minor protein ofapproximately 30 kDa. These proteins appeared to be identical in sizewith the major approximately 70 kDa protein and the minor approximately30 kDa protein produced by B.t. strain EG5144, which are shown in thelane 1 of FIG. 7 and which were prepared in a manner identical to B.t.strain EG7237. This result indicates that the 7.0 kb fragment of pEG272contains two crystal protein genes: one for the approximately 70 kDaprotein and one for the approximately 30 kDa protein.

The gene encoding the approximately 70 kDa protein is the cryIIIC(b)gene, and its encoded protein is the insecticidal CryIIIC(b) protein.The DNA sequence for the cryIIIC(b) gene (SEQ ID NO:1) and the aminoacid sequence for its corresponding deduced protein (SEQ ID NO:2) areshown in FIG. 1.

B.t. strain EG7237 produced approximately three times more 70 kDaprotein, on a weight basis, than did B.t. strain EG5144, as is evidentfrom the protein bands in FIG. 7. Production of the minor 30 kDa proteinin recombinant B.t. strain EG7237 was also increased, as compared withB.t. strain EG5144.

The following Examples 8-11 describe the manner in which theinsecticidal activities of B.t. strain EG5144, B.t. strain EG7237, andthe CryIIIC(b) protein made by these strains were determined.

EXAMPLE 8 Insecticidal Activity of B.t. Strain EG7237 and its CryIIIC(b)Protein Against Southern Corn Rootworm and Colorado Potato Beetle

The insecticidal activity of recombinant B.t. strain EG7237, whichcontains the cryIIIC(b) gene (SEQ ID NO:1) that produces the CryIIIC(b)toxin protein (SEQ ID NO:2), was determined against southern cornrootworm (Diabrotica undecimpunctata howardi) and Colorado potato beetle(Leptinotarsa decemlineata).

For comparison, two other recombinant B.t. strains containingcryIII-type toxin genes in a B.t. strain HD73-26 background were alsoincluded in the bioassay study. These were recombinant B.t. strainEG7235, which contains the cryIIIA gene that produces the CryIIIA toxinprotein, and recombinant B.t. strain EG7225, which contains the cryIIIBgene that produces the CryIIIB toxin protein.

The three B.t. strains were grown in liquid sporulation media at 30° C.until sporulation and cell lysis had occurred. The fermentation brothwas concentrated by microfiltration. The concentrated fermentation brothwas then freeze dried to prepare a B.t. powder suitable for insectbioassay. The amount of CryIII-type toxin protein in each of the B.t.powders was quantified using standard SDS-PAGE techniques.

First instar southern corn rootwom larvae were bioassayed via surfacecontamination of an artificial diet similar to Marrone et al.,J.Econ.Entomol., 78, pp.290-293 (1985), but without formalin. Eachbioassay consisted of eight serial aqueous dilutions with aliquotsapplied to the surface of the diet in a bioassay tray. Each 2 ml well ofthe bioassay tray contained 1 ml diet having a surface area of 175 mm².After the diluent (an aqueous 0.005% Triton® X-100 solution) hadevaporated, the insect larvae were placed on the diet and incubated at28° C. Thirty-two larvae were tested per dose. Mortality was scoredafter 7 days. A control, consisting of diluent only, was also includedin the bioassay study.

First instar Colorado potato beetle larvae were tested using similartechniques, except for the substitution in the artificial diet ofBioServe's No. 9830 insect diet with potato flakes added. Thirty-twolarvae were tested per dose, and mortality was scored at three daysinstead of seven days.

The results of the bioassay study are shown below in Table 3, whereinsecticidal activity is reported as PLC₅₀ values, the concentration ofCryIII-type protein required to kill 50% of the insects tested. Fourreplications per dose were used in the bioassay studies for both insectstested. Data from each of the replicated bioassays were pooled forprobit analysis (R. J. Daum, Bull.Entomol.Soc.Am., 16, pp.10-15 (1970))with mortality corrected for control death, the control being thediluent only (W. S. Abbott, J.Econ.Entomol., 18, pp.265-267 (1925)).Results are shown as the dose amount of CryIII-type protein (in ngCryIII protein per mm² of diet surface) resulting in PLC⁵⁰. Confidenceintervals, at 95%, are given within parentheses below the PLC50 values.

                                      TABLE 3                                     __________________________________________________________________________    Insecticidal Activity of Recombinant B.t. Strains EG7237, EG7235 and          EG7225                                                                                       CryIII Protein                                                                        Southern Corn Rootworm                                                                     Colorado Potato Beetle                                   Concentration                                                                         PLC.sub.50   PLC.sub.50                                B.t. Strain                                                                          CryIII Protein                                                                        (%)     (ng CryIII protein/mm.sup.2)                                                               (ng CryIII protein/mm.sup.2)              __________________________________________________________________________    B.t. EG7237                                                                          CryIIIC(b)                                                                            7.2     1548         6.92                                                             (1243-1992)  (5.15-9.10)                               B.t. EG7235                                                                          CryIIIA 28.4    6% control   0.34                                                             at 4570      (0.30-0.39)                               B.t. EG7225                                                                          CryIIIB 9.4     20% control  1.26                                                             at 4570      (1.07-1.46)                               __________________________________________________________________________

The results of this bioassay study demonstrate that B.t. strain EG7237which produces the CryIIIC(b) toxin protein (SEQ ID NO:2) isinsecticidal to southern corn rootworm. In contrast, the CryIIIA andCryIIIB toxin proteins of B.t. strains EG7235 and EG7225, respectively,appear to have no measurable activity against this insect at the highestdose level tested.

All three of the B.t. strains exhibit insecticidal activity againstColorado potato beetle larvae, with the CryIIIA toxin protein of B.t.strain EG7235 being significantly more potent than the CryIIIC(b) toxinprotein (SEQ ID NO:2) of B.t. strain EG7237 and with the CryIIIB toxinprotein of B.t. strain EG7225 having insecticidal activity fallingbetween that shown for CryIIIA and CryIIIC(b).

These results suggest that the insecticidal activity of specificCryIII-type toxin proteins varies for different insect genera within theorder Coleoptera.

EXAMPLE 9 Insecticidal Activity of B.t. Strain EG7237 and its CryIIIC(b)Protein Against Mexican Bean Beetle

The insecticidal activity of recombinant B.t. strain EG7237, evaluatedin Example 8, was also determined against Mexican bean beetle (Epilachnavarivestis). As in Example 8, recombinant B.t. strains EG7235 and EG7225were included for comparison, and all B.t. powders were prepared as inExample 8.

First instar Mexican bean beetle larvae were bioassayed by a leaf dipprocedure, since a suitable artificial diet is not available for thisinsect. Soybean leaves were dipped into known treatment concentrationsof the B.t. powder suspended in an aqueous 0.1% Triton® X-100 solution.After excess material had dripped off, the leaves were allowed to dry.Leaves dipped in 0.1% Triton® X-100 served as untreated controls. Twentyinsect larvae were confined to a petri dish with treated leaves,incubated at 25° C. and allowed to feed for three days, at which timemortality was scored.

The results of the bioassay study are shown below in Table 4, whereinsecticidal activity is reported as PLC₅₀ values, the concentration ofCryIII-type protein required to kill 50% of the insects tested. The datawere handled as described in Example 8, for Table 3. Results are shownas the dose amount of CryIII-type protein (in mg CryIII protein/mlsolution used in the leaf dip) resulting in PLC₅₀. Confidence intervals,at 95%, are given within parentheses following the PLC₅₀ values.

                  TABLE 4                                                         ______________________________________                                        Insecticidal Activity of B.t. Strains EG7237, EG7235 and                      EG7225 Against Mexican Bean Beetle                                                    CryIII    No. of     PLC.sub.50                                       B.t. Strain                                                                           Protein   Replications                                                                             (mg CryIIIprotein/ml)                            ______________________________________                                        B.t.    CryIIIC(b)                                                                              4          4.2 (2.5-6.5)                                    EG7237                                                                        B.t.    CryIIIA   4          16% control at 60                                EG7235                                                                        B.t.    CryIIIB   4          51.8 (31-209)                                    EG7225                                                                        ______________________________________                                    

The results of this bioassay study demonstrate that B.t. strain EG7237which produces the CryIIIC(b) toxin protein (SEQ ID NO:2) issignificantly more insecticidal to Mexican bean beetle than theCryIIIB-producing B.t. strain EG7225. B.t. strain EG7235 which producesCryIIIA toxin protein exhibited no measurable insecticidal activity atthe highest dose tested.

These results are further evidence that the insecticidal activity ofspecific CryIII-type toxin proteins varies widely for insect generawithin the order Coleoptera.

EXAMPLE 10 Insecticidal Activity of B.t. Strain EG5144 Against SouthernCorn Rootworm

The insecticidal activity of B.t. strain EG5144 was evaluated againstSouthern corn rootworm (Diabrotica undecimpunctata howardi). Forcomparison, B.t. strain EG4961 which produces the CryIIIC(a) toxinprotein was included in the bioassay study.

The bioassay procedure for southern corn rootworm in this Exampledetermined PLC₅₀ values, the concentration of CryIII-type proteinrequired to kill 50% of the insects tested. The procedure was similar tothe artificial diet bioassay carried out in the previous Example, usingthirty-two first instar southern corn rootworm larvae per dose. Datafrom each of the replicated bioassays were pooled for probit analysis(R. J. Daum, Bull.Entomol.Soc.Am., 16, pp.10-15 (1970)) with mortalitycorrected for control death, the control being the diluent only (W. S.Abbott, J.Econ.Entomol., 18, pp.265-267 (1925)). Results are reportedfor two separate tests as the dose amount of CryIII-type protein (ngCryIII protein per mm² of diet surface) resulting in PLC₅₀. Confidenceintervals, at 95%, are given within parentheses following the PLC₅₀values. In Test 1 four replications per dose were used, and in Test 2,carried out at a later date, two replications were used.

The B.t. strains used in this Example were prepared as described for theB.t. strains in Example 8, except that the fermentation broth wasconcentrated by centrifugation.

The results of this bioassay study with southern corn rootworm are shownbelow in Table 5.

                  TABLE 5                                                         ______________________________________                                        Insecticidal Activity of B.t. Strains EG5144                                  and EG4961 Against Southern Corn Rootworm                                                          CryIII Protein                                                                            PLC.sub.50                                                        Concentration                                                                             (ng CryIII                                   B.t. Strain                                                                            CryIII Protein                                                                            (%)         protein/mm.sup.2)                            ______________________________________                                        B.t. EG5144                                                                            CryIIIC(b)  Test 1: 4.0   944 (690-1412)                                                  Test 2: 6.4   1145 (773-2185)                            B.t. EG4961                                                                            CryIIIC(a)  Test 1: 11.6  102 (86-119)                                                    Test 2: 11.6  165 (121-220)                              ______________________________________                                         This bioassay study demonstrates that both B.t. strain EG5144 and B.t.     strain EG4961, which produce CryIIIC-type proteins, provide quantifiable     insecticidal activity against southern corn rootworm.

EXAMPLE 11 Insecticidal Activity of B.t. Strain EG5144 Against JapaneseBeetle Larvae

The insecticidal activity of B.t. strain EG5144 was evaluated againstJapanese beetle larvae, also known as white grubs (Popillia japonica).For comparison, B.t. strain EG4961 which produces the CryIIIC(a) toxinprotein was included in the bioassay study, as were B.t. strain EG2158which produces the CryIIIA toxin protein and B.t. strain EG2838 whichproduces the CryIIIB toxin protein.

The bioassay procedure in this Example was a screening assay, at asingle dose of CryIII-type protein in a diet incorporation assay (1 mgCryIII-type protein per ml diet). B.t. powder to be tested, suspended ina diluent (an aqueous 0.005% Triton® X-100 solution) was incorporatedinto 100 ml of hot (50°-60° C.), liquid artificial diet (based on theinsect diet described by Ladd, Jr. in J.Econ.Entomol., 79, pp.668-671(1986)). The mixture was allowed to solidify in petri dishes, and one 19mm diameter plug of this material then placed in each well of a plasticice cube tray. One grub was introduced per well of the trays, the wellswere covered with moist germination paper overlaid with aluminum foil,and the trays were held at 25° C. for seven days before mortality wasscored. The insects tested were third instar Japanese beetle grubs. Tworeplications of sixteen insects each were carried out in this study.

The results of this screening bioassay study are shown below in Table 6,where insecticidal activity is reported as percentage insect mortality,with the mortality being corrected for control death, the control beingdiluent only incorporated into the diet plug. Results were obtained at asingle dose rate of CryIII-type protein: 1 mg CryIII-type protein per mlof diet; percentage CryIII-type protein present in the respective B.t.powders is also shown in Table 6.

                                      TABLE 6                                     __________________________________________________________________________    Insecticidal activity of B.t. Strains EG5144, EG4961, EG2158 and EG2838       Against Japanese Beetle Grubs                                                                CryIII-type Protein                                                                     CryIII-type Protein Dose                                                                   Insect                                                 in B.t. Powder                                                                          (mg CryIII-type                                                                            Mortality                               B.t. Strain                                                                          CryIII Protein                                                                        (wt. %)   protein/ml diet)                                                                           (%)                                     __________________________________________________________________________    B.t. EG5144                                                                          CryIIIC(b)                                                                            5.4       1            62.5                                    B.t. EG4961                                                                          CryIIIC(a)                                                                            18.0      1            9                                       B.t. EG2158                                                                          CryIIIA 14.0      1            44                                      B.t. EG2838                                                                          CryIIIB 7.2       1            48                                      __________________________________________________________________________

The insecticidal performance against Japanese beetle grubs of B.t.strain EG5144 with its CryIIIC(b) toxin protein (SEQ ID NO:2) is clearlysuperior to that of B.t. strain EG4961 with its CryIIIC(a) protein.

With respect to B.t. strains EG2158 and B.t. strain EG2838, B.t. strainEG5144 exhibited superior insecticidal performance against Japanesebeetle grubs.

B.t. strain EG5145, whose characteristics are similar to those of B.t.strain EG5144, has been found to exhibit insecticidal activity againstJapanese beetle grubs equivalent to that of B.t. strain EG5144, althoughthe bioassay data are not presented in this Example 11.

Microorganism Deposits

To assure the availability of materials to those interested members ofthe public upon issuance of a patent on the present application,deposits of the following microorganisms were made prior to the filingof present application with the ARS Patent Collection, AgriculturalResearch Culture Collection, Northern Regional Research Laboratory(NRRL), 1815 North University Street, Peoria, Ill. 61604, as indicatedin the following Table 7:

                  TABLE 7                                                         ______________________________________                                        Bacterial Strain                                                                         NRRL Accession No.                                                                           Date of Deposit                                     ______________________________________                                        B.t. EG2158                                                                              B-18213        April 29, 1987                                      B.t. HD73-26                                                                             B-18508        June 12, 1989                                       B.t. EG2838                                                                              B-18603        February 8, 1990                                    B.t. EG5144                                                                              B-18655        May 22, 1990                                        B.t. EG7237                                                                              B-18736        October 17, 1990                                    E. coli EG7236                                                                           B-18662        June 6, 1990                                        B.t. EG5145                                                                              B-18920        November 21, 1991                                   ______________________________________                                    

These microorganism deposits were made under the provisions of the"Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure". All restrictionson the availability to the public of these deposited microorganisms willbe irrevocably removed upon issuance of a United States patent based onthis application.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification as indicating the scope of theinvention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2430 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 144 . . . 2099                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCATATACAACTTATCAGGAAGGGGGGGATGCACAAAGAAGAAAAGAATAAGAAGTGAAT60                GTTTATAATGTTCAATAGTTTTATGGGAAGGCATTTTATCAGGTAGAAAGTTATGTATTA120               TGATAAGAATG GGAGGAAGAAAAATGAATCCAAACAATCGAAGTGAACAT170                        MetAsnProAsnAsnArgSerGluHis                                                   15                                                                            GATACGATAAAGGTTACACCTA ACAGTGAATTGCCAACTAACCATAAT218                          AspThrIleLysValThrProAsnSerGluLeuProThrAsnHisAsn                              10152025                                                                      CAATATCCTTTAGCTGACAA TCCAAATTCGACACTAGAAGAATTAAAT266                          GlnTyrProLeuAlaAspAsnProAsnSerThrLeuGluGluLeuAsn                              303540                                                                        TATAAAGAATTTTTAAGAATG ACTGAAGACAGTTCTACGGAAGTGCTA314                          TyrLysGluPheLeuArgMetThrGluAspSerSerThrGluValLeu                              455055                                                                        GACAACTCTACAGTAAAAGATGCA GTTGGGACAGGAATTTCTGTTGTA362                          AspAsnSerThrValLysAspAlaValGlyThrGlyIleSerValVal                              606570                                                                        GGGCAGATTTTAGGTGTTGTAGGAGTTCCAT TTGCTGGGGCACTCACT410                          GlyGlnIleLeuGlyValValGlyValProPheAlaGlyAlaLeuThr                              758085                                                                        TCATTTTATCAATCATTTCTTGACACTATATGGCCAAGTGA TGCTGAC458                          SerPheTyrGlnSerPheLeuAspThrIleTrpProSerAspAlaAsp                              9095100105                                                                    CCATGGAAGGCTTTTATGGCACAAGTTGAAGTACTGATA GATAAGAAA506                          ProTrpLysAlaPheMetAlaGlnValGluValLeuIleAspLysLys                              110115120                                                                     ATAGAGGAGTATGCTAAAAGTAAAGCTCTTGCAGAGTTA CAGGGTCTT554                          IleGluGluTyrAlaLysSerLysAlaLeuAlaGluLeuGlnGlyLeu                              125130135                                                                     CAAAATAATTTCGAAGATTATGTTAATGCGTTAAATTCCTGGA AGAAA602                          GlnAsnAsnPheGluAspTyrValAsnAlaLeuAsnSerTrpLysLys                              140145150                                                                     ACACCTTTAAGTTTGCGAAGTAAAAGAAGCCAAGATCGAATAAGGGAA 650                          ThrProLeuSerLeuArgSerLysArgSerGlnAspArgIleArgGlu                              155160165                                                                     CTTTTTTCTCAAGCAGAAAGTCATTTTCGTAATTCCATGCCGTCATTT698                           LeuPhe SerGlnAlaGluSerHisPheArgAsnSerMetProSerPhe                             170175180185                                                                  GCAGTTTCCAAATTCGAAGTGCTGTTTCTACCAACATATGCACAAGCT746                           Ala ValSerLysPheGluValLeuPheLeuProThrTyrAlaGlnAla                             190195200                                                                     GCAAATACACATTTATTGCTATTAAAAGATGCTCAAGTTTTTGGAGAA794                           AlaA snThrHisLeuLeuLeuLeuLysAspAlaGlnValPheGlyGlu                             205210215                                                                     GAATGGGGATATTCTTCAGAAGATGTTGCTGAATTTTATCATAGACAA842                           GluTrpGl yTyrSerSerGluAspValAlaGluPheTyrHisArgGln                             220225230                                                                     TTAAAACTTACGCAACAATACACTGACCATTGTGTCAATTGGTATAAT890                           LeuLysLeuThrGln GlnTyrThrAspHisCysValAsnTrpTyrAsn                             235240245                                                                     GTTGGATTAAATGGTTTAAGAGGTTCAACTTATGATGCATGGGTCAAA938                           ValGlyLeuAsnGlyLeuArgGly SerThrTyrAspAlaTrpValLys                             250255260265                                                                  TTTAACCGTTTTCGCAGAGAAATGACTTTAACTGTATTAGATCTAATT986                           PheAsnArgPheArgArgGluM etThrLeuThrValLeuAspLeuIle                             270275280                                                                     GTACTTTTCCCATTTTATGATGTTCGGTTATACTCAAAAGGTGTTAAA1034                          ValLeuPheProPheTyrAspVa lArgLeuTyrSerLysGlyValLys                             285290295                                                                     ACAGAACTAACAAGAGACATTTTTACGGATCCAATTTTTTCACTCAAT1082                          ThrGluLeuThrArgAspIlePheThr AspProIlePheSerLeuAsn                             300305310                                                                     ACTCTTCAGGAGTATGGACCAACTTTTTTGAGTATAGAAAACTCTATT1130                          ThrLeuGlnGluTyrGlyProThrPheLeuSer IleGluAsnSerIle                             315320325                                                                     CGAAAACCTCATTTATTTGATTATTTACAGGGTATTGAATTTCATACG1178                          ArgLysProHisLeuPheAspTyrLeuGlnGlyIleGluPheH isThr                             330335340345                                                                  CGTCTTCAACCTGGTTACTCTGGGAAAGATTCTTTCAATTATTGGTCT1226                          ArgLeuGlnProGlyTyrSerGlyLysAspSerPheAsnTy rTrpSer                             350355360                                                                     GGTAATTATGTAGAAACTAGACCTAGTATAGGATCTAGTAAGACAATT1274                          GlyAsnTyrValGluThrArgProSerIleGlySerSerLys ThrIle                             365370375                                                                     ACTTCCCCATTTTATGGAGATAAATCTACTGAACCTGTACAAAAGTTC1322                          ThrSerProPheTyrGlyAspLysSerThrGluProValGlnLys Leu                             380385390                                                                     AGCTTTGATGGACAAAAAGTTTATCGAACTATAGCTAATACAGACGTA1370                          SerPheAspGlyGlnLysValTyrArgThrIleAlaAsnThrAspVal                               395400405                                                                    GCGGCTTGGCCGAATGGCAAGATATATTTTGGTGTTACGAAAGTTGAT1418                          AlaAlaTrpProAsnGlyLysIlrTyrPheGlyValThrLysValAsp                              410 415420425                                                                 TTTAGTCAATATGATGATCAAAAAAATGAAACTAGTACACAAACATAT1466                          PheSerGlnTyrAspAspGlnLysAsnGluThrSerThrGlnThrTyr                               430435440                                                                    GATTCAAAAAGAAACAATGGCCATGTAGGTGCACAGGATTCTATTGAC1514                          AspSerLysArgAsnAsnGlyHisValGlyAlaGlnAspSerIleAsp                              445 450455                                                                    CAATTACCACCAGAAACAACAGATGAACCACTTGAAAAAGCATATAGT1562                          GlnLeuProProGluThrThrAspGluProLeuGluLysAlaTyrSer                              460 465470                                                                    CATCAGCTTAATTACGCGGAATGTTTCTTAATGCAGGACCGTCGTGGA1610                          HisGlnLeuAsnTyrAlaGluCysPheLeuMetGlnAspArgArgGly                              475480 485                                                                    ACAATTCCATTTTTTACTTGGACACATAGAAGTGTAGACTTTTTTAAT1658                          ThrIleProPhePheThrTrpThrHisArgSerValAspPhePheAsn                              49049550 0505                                                                 ACAATTGATGCTGAAAAGATTACTCAACTTCCAGTAGTGAAAGCATAT1706                          ThrIleAspAlaGluLysIleThrGlnLeuProValValLysAlaTyr                              510515 520                                                                    GCCTTGTCTTCAGGTGCTTCCATTATTGAAGGTCCAGGATTCACAGGA1754                          AlaLeuSerSerGlyAlaSerIleIleGluGlyProGlyPheThrGly                              525530 535                                                                    GGAAATTTACTATTCCTAAAAGAATCTAGTAATTCAAATGCTAAATTT1802                          GlyAsnLeuLeuPheLeuLysGluSerSerAsnSerIleAlaLysPhe                              540545 550                                                                    AAAGTTACATTAAATTCAGCAGCCTTGTTACAACGATATCGTGTAAGA1850                          LysValThrLeuAsnSerAlaAlaLeuLeuGlnArgTyrArgValArg                              555560565                                                                     ATA CGCTATGCTTCTACCACTAACTTACGACTTTTTGTGCAAAATTCA1898                         IleArgTyrAlaSerThrThrAsnLeuArgLeuPheValGlnAsnSer                              570575580585                                                                  A ACAATGATTTTATTGTCATCTACATTAATAAAACTATGAATATAGAT1946                         AsnAsnAspPheIleValIleTyrIleAsnLysThrMetAsnIleAsp                              590595600                                                                     GA TGATTTAACATATCAAACATTTGATCTCGCAACTACTAATTCTAAT1994                         AspAspLeuThrTyrGlnThrPheAspLeuAlaThrThrAsnSerAsn                              605610615                                                                     ATGGGG TTCTCGGGTGATACGAATGAACTTATAATAGGAGCAGAATCT2042                         MetGlyPheSerGlyAspThrAsnGluLeuIleIleGlyAlaGluSer                              620625630                                                                     TTCGTTTCTAAT GAAAAAATCTATATAGATAAGATAGAATTTATCCCA2090                         PheValSerAsnGluLysIleTyrIleAspLysIleGluPheIlePro                              635640645                                                                     GTACAATTGTAAGGAGATTTTGAAA TGTAGGGCGATGGTCAAAATGAAA2139                        ValGlnLeu                                                                     650                                                                           GAATAGGAAGGTGAATTTTGATGGTTAGGAAACATTCTTTTAAGAAAAGCAACATGGAAA2199              AGTATACAGTACAAATATTAGAAATAAAATTTATTAACACAGGGGAAGATGGTAAACCAG 2259             AACCGTATGGTTATATTGACTTTTATTATCAACCTGCTCCTAACCTGAGAGAAGAAAAAG2319              TAAGAATTTGGGAAGAGAAAAATAGTAGCTCTCCACCTTCAATAGAAGTTATTACGGGGC2379              TAACTTTTAATATCATGGCTACTTCACTTAGCCGATTATGTT TTGAAGGTT2430                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 652 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAsnProAsnAsnArgSerGluHisAspThrIle LysValThrPro                             151015                                                                        AsnSerGluLeuProThrAsnHisAsnGlnTyrProLeuAlaAspAsn                              202530                                                                        ProAsnSerThrLeuGluGluLeuAsnTyrLysGluPheLeuArgMet                              354045                                                                        ThrGluAspSerSerThrGluValLeuAspAsnSerThrValLysAsp                              50 5560                                                                       AlaValGlyThrGlyIleSerValValGlyGlnIleLeuGlyValVal                              65707580                                                                      GlyValProPheAlaGlyAlaLeuT hrSerPheTyrGlnSerPheLeu                             859095                                                                        AspThrIleTrpProSerAspAlaAspProTrpLysAlaPheMetAla                              100105 110                                                                    GlnValGluValLeuIleAspLysLysIleGluGluTyrAlaLysSer                              115120125                                                                     LysAlaLeuAlaGluLeuGlnGlyLeuGlnAsnAsnPheGluAspTyr                               130135140                                                                    ValAsnAlaLeuAsnSerTrpLysLysThrProLeuSerLeuArgSer                              145150155160                                                                  LysArgSerGlnAs pArgIleArgGluLeuPheSerGlnAlaGluSer                             165170175                                                                     HisPheArgAsnSerMetProSerPheAlaValSerLysPheGluVal                              180 185190                                                                    LeuPheLeuProThrTyrAlaGlnAlaAlaAsnThrHisLeuLeuLeu                              195200205                                                                     LeuLysAspAlaGlnValPheGlyGluGluTrpGlyT yrSerSerGlu                             210215220                                                                     AspValAlaGluPheTyrHisArgGlnLeuLysLeuThrGlnGlnTyr                              225230235240                                                                  Thr AspHisCysValAsnTrpTyrAsnValGlyLeuAsnGlyLeuArg                             245250255                                                                     GlySerThrTyrAspAlaTrpValLysPheAsnArgPheArgArgGlu                               260265270                                                                    MetThrLeuThrValLeuAspLeuIleValLeuPheProPheTyrAsp                              275280285                                                                     ValArgLeuTyrSerLysGlyValLy sThrGluLeuThrArgAspIle                             290295300                                                                     PheThrAspProIlePheSerLeuAsnThrLeuGlnGluTyrGlyPro                              305310315 320                                                                 ThrPheLeuSerIleGluAsnSerIleArgLysProHisLeuPheAsp                              325330335                                                                     TyrLeuGlnGlyIleGluPheHisThrArgLeuGlnProGlyTyrS er                             340345350                                                                     GlyLysAspSerPheAsnTyrTrpSerGlyAsnTyrValGluThrArg                              355360365                                                                     ProSerIleGlySer SerLysThrIleThrSerProPheTyrGlyAsp                             370375380                                                                     LysSerThrGluProValGlnLysLeuSerPheAspGlyGlnLysVal                              385390 395400                                                                 TyrArgThrIleAlaAsnThrAspValAlaAlaTrpProAsnGlyLys                              405410415                                                                     IleTyrPheGlyValThrLysValAspPheSerGl nTyrAspAspGln                             420425430                                                                     LysAsnGluThrSerThrGlnThrTyrAspSerLysArgAsnAsnGly                              435440445                                                                     His ValGlyAlaGlnAspSerIleAspGlnLeuProProGluThrThr                             450455460                                                                     AspGluProLeuGluLysAlaTyrSerHisGlnLeuAsnTyrAlaGlu                              465470 475480                                                                 CysPheLeuMetGlnAspArgArgGlyThrIleProPhePheThrTrp                              485490495                                                                     ThrHisArgSerValAspPhePhe AsnThrIleAspAlaGluLysIle                             500505510                                                                     ThrGlnLeuProValValLysAlaTyrAlaLeuSerSerGlyAlaSer                              515520 525                                                                    IleIleGluGlyProGlyPheThrGlyGlyAsnLeuLeuPheLeuLys                              530535540                                                                     GluSerSerAsnSerIleAlaLysPheLysValThrLeuAsnSerAla                              545 550555560                                                                 AlaLeuLeuGlnArgTyrArgValArgIleArgTyrAlaSerThrThr                              565570575                                                                     AsnLeuArgLeu PheValGlnAsnSerAsnAsnAspPheIleValIle                             580585590                                                                     TyrIleAsnLysThrMetAsnIleAspAspAspLeuThrTyrGlnThr                              595 600605                                                                    PheAspLeuAlaThrThrAsnSerAsnMetGlyPheSerGlyAspThr                              610615620                                                                     AsnGluLeuIleIleGlyAlaGluSerPheValSerAsnGlyLys Ile                             625630635640                                                                  TyrIleAspLysIleGluPheIleProValGlnLeu                                          645650                                                                    

We claim:
 1. An isolated coleopteran-toxic protein having the amino acidsequence illustrated in FIG. 1 (SEQ ID NO: 2).
 2. A coleopteran-toxicprotein encoded by an isolated gene having a coding region extendingfrom nucleotide bases 144 to 2099 in the nucleotide base sequenceillustrated in FIG. 1 (SEQ ID NO: 1).
 3. An insecticide compositioncomprising the protein of claim 1 or 2 and an agriculturally acceptablecarrier.
 4. An insecticide composition comprising the coleopteran-toxicprotein of claim 1, in combination with an agriculturally acceptablecarrier.
 5. The insecticide composition of claim 4 wherein thecoleopteran-toxic protein is associated with a Bacillus thuringiensisbacterium which has produced such protein.
 6. An insecticide compositioncomprising the coleopteran-toxic protein identical to that from abiologically pure culture of a Bacillus thuringiensis bacterium selectedfrom the group consisting of a culture deposited with the NRRL havingaccession number NRRL B-18655 and NRRL B-18920, in combination with anagriculturally acceptable carrier.